An estimated 3% of the world's population is seropositive for hepatitis C virus (HCV) (1, 2, 3). Approximately 70% of seropositive individuals develop a chronic infection. Infection with HCV predisposes victims to liver pathology, including fibrosis, cirrhosis, and hepatocellular carcinoma (18). Most seropositive persons eventually develop hepatocellular carcinoma (4), and therefore HCV infection is also the leading cause of liver failure and the need for liver transplants in the U.S. (3,5).
HCV is a 9.6 kb positive strand RNA virus of the Flavirviradae family, genus Hepacivirus (6). The RNA comprises a 5′ UTR (untranslated region) of approximately 340 nucleotides that includes an internal ribosome entry sequence (IRES), a single open reading frame (ORF) of approximately 9000 nucleotides and a 3′ UTR of approximately 230 nucleotides. The internal ribosome entry sequence mediates initiation of viral RNA. The single open reading frame is translated into a polyprotein of approximately 3000 amino acid residues. This is cleaved by proteases to produce at least three structural proteins (core, E1, and E2) and six non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a, and NS5b) (3).
The core protein forms a capsid, and E1 and E2 interact with plasma membranes of hepatocytes. NS2 is a zinc metaloprotease that cleaves the polyprotein at the NS2-NS3 junction between Leu1026 and Ala1027 (7). NS3 is a bifunctional enzyme, with its N terminus a serine protease that cleaves the rest of the polyprotein in conjunction with its cofactor, NS4a. The C terminus of NS3 is a helicase that is responsible for unwinding and separating putative double-stranded replication intermediates in the HCV life cycle (3,8). The roles of NS4b and NS5a have not been well defined, although it is postulated that NS5a may act as an interferon antagonist. NS5b is an RNA-dependent RNA polymerase that can copy the positive and negative strands of RNA.
Recently another ORF of HCV has been identified, which encodes protein F of unknown function (9).
NS3 is a helicase. Helicases are enzymes that unwind dsDNA and dsRNA in various biological processes, including replication, recombination, and repair. Helicases act by converting the chemical energy of ATP hydrolysis to the mechanical energy of unwinding. NS3 is a 67 kDa, 3′-to-5′ RNA-DNA helicase, of the SFII superfamily, and is thought to unwind dsRNA and other secondary structures during HCV replication (11). The oligomeric state of NS3 has been a subject of debate, since it has been shown to be a monomer (12-14), dimer (15), and oligomer (16) in the literature.
The current treatments for HCV infection are alpha interferon (IFN-α) in combination with ribavirin or a polyethylene glycol-modified form of IFN-α. But sustained responses are only observed in about half of the treated patients, and effectiveness varies depending on the HCV genotype (Blight, K. J. et al. 2002. J. Virol. 76:13001). Thus, improved treatments for HCV infection are needed. Treatments for HCV infection would include methods of inhibiting HCV replication. Thus, compounds and methods for inhibiting HCV replication are needed. Methods of identifying compounds that inhibit HCV replication are also needed.